【作者】 戴毅敏；

【导师】 胡娅莉；

【作者基本信息】 南京大学 ， 外科学， 2011， 博士

【摘要】 目的研究在LPS诱导的天然免疫反应中,人绒毛外滋养细胞HTR-8/SVneo中BIC/miR-155表达上调的可能机制及其对细胞功能的影响,以探讨miR-155在重度PE发病中的作用。方法用含不同浓度LPS (0-800ng/ml)的培养液培养HTR-8/SVneo细胞,检测miR-155相对表达量及转录因子AP-1(JunB. FosB亚单位)和NF-kB (p-P65. P65亚单位)的变化情况。根据BIC/miR-155启动子上游转录因子结合位点序列,构建含AP-1、 NF-kB1和NF-kB2及相应位点突变的不同组合的启动子,应用qRT-PCR.荧光素酶报告基因系统、诱骗寡核苷酸技术和DNA免疫沉淀法评估启动子活性以及与相应DNA序列特异性结合的活性。甲基化敏感性限制性内切酶-半定量PCR法分析启动子甲基化水平。通过质粒转染、划痕试验、光镜下计数的方法,检测转染miR-155或anti-miR-155对滋养细胞迁移、合体化功能的影响。结果在HTR-8/SVneo中,低浓度LPS上调JunB/FosB (25-100ng/ml), NF-kB p-P65/P65(50-200ng/ml), miR-155相对表达量呈浓度、时间依赖性增加约4倍。含AP-1结合位点的BIC/miR-155启动子活性显著高于含NF-kB1和/或NF-kB2启动子；20mM SP600125、50mM PDTC或20mM SP600125联合50mM PDTC预处理后,miR-155相对表达量均显著下降(P<0.05)。单独转染NF-kB1或/和NF-KB2诱骗寡核苷酸,miR-155表达量无显著变化(P>0.05)；同时转染AP-1NF-kB1和NF-kB2三种不同序列的诱骗寡核苷酸,或单独转染AP-1诱骗寡核苷酸,miR-155表达量均显著降低(P<0.05)。和正常妊娠相比,miR-155高表达的PE胎盘中,JunB/FosB, NF-kB p65的表达显著增加。在体外,0.1μg/ml LPS培养HTR-8/SVneo后,BIC/miR-155启动子甲基化水平是对照组的57%；1.0μg/ml LPS作用下,甲基化水平比对照组上升21%,差异均有统计学意义(P<0.05)。转染miR-155质粒的HTR-8/SVneo在24h和32h的细胞迁移率分别为对照组的66.1%和53.6%,差异显著(P<0.05)；转染anti-miR-155的细胞中,24h和32h的细胞迁移率分别是对照组的2.0倍和1.9倍,差异显著(P<0.05)。转染miR-155后,合体细胞样的多核滋养细胞总数(2-5个核/细胞)占总细胞数的比例显著增加(P<0.05),同时,包膜蛋白syncytin1和CD81的相对表达量分别是对照组的5倍和8倍(P<0.05)；相反,转染anti-miR-155的细胞中,合体细胞样的多核滋养细胞总数占总细胞数的比例显著减少(P<0.05)。结论由低剂量LPS诱导的炎症反应可激活人绒毛外滋养细胞HTR-8/SVneo中的AP-1和NF-kB通路、上调并募集转录因子JunB/FosB, NF-kB p-P65与BIC/miR-155启动子上相应的DNA序列特异性结合,转录BIC/miR-155,通过抑制滋养细胞迁移、促进其合体化形成,参与重度PE的发病。在该信号通路上,保守的AP-1结合位点是BIC/miR-155转录激活中的关键元件。更多还原

【Abstract】 Objective A low dose injection of lipopolysaccharides (LPS) may induce pre-eclampsia (PE)-like symptoms in rats, and microRNA-155(miR-155) is elevated in the placentas of patients with PE. Our goal was to investigate the association of miR-155with PE and the pathways involved using human-trophoblast-derived HTR-8/SVneo cells stimulated with LPS.Methods We measured miR-155in HTR-8/SVneo cells treated with LPS (25-800ng/ml) using real-time PCR. Western blotting was used to study transcription factor activated protein1(AP-1)(JunB and FosB subunits) and nuclear factor (NF)-kB p65in the HTR-8/SVneo cells and placentas from patients with PE. DNA precipitation assays and luciferase reporter analysis were used to evaluate the regulation of miR-155by AP-1and NF-kB. Cell migration was determined by scratch assay. Syncytialization of HTR-8/SVneo cells was analysed following transfection with miR-155.Results miR-155was increased together with AP-1and NF-kB in HTR-8/SVneo cells incubated with low dose of LPS (≤100ng/ml; P<0.05versus baseline). Both JunB/FosB and p65were increased in placenta from women with severe PE versus a normal pregnancy, with elevated expression of miR-155(P<0.05). For specific DNA-binding sites upstream of BIC/miR-155gene promoter, the AP-1site was more important than the NF-kB site for increasing miR-155in HTR-8/SVneo cells. The cells with enforced expression of miR-155showed a reduced ability to migrate (P<0.05) and an increased number of syncytiotrophoblast-like multinuclear cells (P<0.05).Conclusions LPS may induce remodelling of the human-trophoblast-derived HTR-8/SVneo cells by increasing miR-155, acting in part through the AP-1and NF-kB pathways.更多还原